fig8: MADD-2 promotes stable UNC-40–mediated F-actin polarization. Anterior is left; ventral is down. (A) Time series shows F-actin stably polarized at the basal surface of the AC of a wild-type animal. (B) The volume of the stable basal F-actin patch (green line). (C) Similar localization in four other cases. (D) The time series shows multiple mislocalized F-actin patches in the AC of a madd-2 mutant. (E) The volume of individual F-actin patches in a madd-2 mutant. Green lines represent basal patches, and purple lines represent mislocalized patches. (F) Similar analysis of four additional madd-2 mutants. Large mislocalized F-actin patches were observed in 50% of madd-2 mutants (n = 6/12) but were not observed in any wild-type animals (n = 10/10). Isosurface thresholds were set to a stringent level that delineated ectopic and basal patches in madd-2 animals to evaluate the AC’s ability to polarize F-actin at the invasive membrane toward endogenous UNC-6 (see Materials and methods). This thresholding resulted in a reduction of reported F-actin volume compared with previous figures. All animals were examined at the P6.p two-cell stage. Bars, 5 µm.

Mentions:
The interplay of positive and negative feedback is important in robust polarization responses in yeast (Howell et al., 2012; Bendezú and Martin, 2013; Dyer et al., 2013; Wu and Lew, 2013). Given that MADD-2 regulates this balance with UNC-40 clustering, we hypothesized that madd-2 mutants may show defects in UNC-40 polarization toward UNC-6. To test this idea, we examined F-actin and UNC-40 polarization toward endogenous UNC-6 in madd-2 mutants. Notably, UNC-40 and F-actin were no longer tightly localized to the invasive cell membrane of the AC in madd-2 mutants (Fig. 7, F and G). Furthermore, in contrast to the robust localization of F-actin along the invasive cell membrane in wild-type animals (n = 10/10 ACs; Fig. 8, A–C; and Video 5), time-lapse analyses revealed that madd-2 mutants often had transient, mislocalized patches of F-actin along the AC’s apical and lateral domains (n = 6/12 animals; Fig. 8, D–F; and Video 5). The specific function of MADD-2 in regulating UNC-6–independent UNC-40 clustering and the perturbation in UNC-40 polarization toward UNC-6 in madd-2 mutants offer compelling evidence that ligand-independent UNC-40 (DCC) clustering is required for robust polarization toward UNC-6 (netrin).

fig8: MADD-2 promotes stable UNC-40–mediated F-actin polarization. Anterior is left; ventral is down. (A) Time series shows F-actin stably polarized at the basal surface of the AC of a wild-type animal. (B) The volume of the stable basal F-actin patch (green line). (C) Similar localization in four other cases. (D) The time series shows multiple mislocalized F-actin patches in the AC of a madd-2 mutant. (E) The volume of individual F-actin patches in a madd-2 mutant. Green lines represent basal patches, and purple lines represent mislocalized patches. (F) Similar analysis of four additional madd-2 mutants. Large mislocalized F-actin patches were observed in 50% of madd-2 mutants (n = 6/12) but were not observed in any wild-type animals (n = 10/10). Isosurface thresholds were set to a stringent level that delineated ectopic and basal patches in madd-2 animals to evaluate the AC’s ability to polarize F-actin at the invasive membrane toward endogenous UNC-6 (see Materials and methods). This thresholding resulted in a reduction of reported F-actin volume compared with previous figures. All animals were examined at the P6.p two-cell stage. Bars, 5 µm.

Mentions:
The interplay of positive and negative feedback is important in robust polarization responses in yeast (Howell et al., 2012; Bendezú and Martin, 2013; Dyer et al., 2013; Wu and Lew, 2013). Given that MADD-2 regulates this balance with UNC-40 clustering, we hypothesized that madd-2 mutants may show defects in UNC-40 polarization toward UNC-6. To test this idea, we examined F-actin and UNC-40 polarization toward endogenous UNC-6 in madd-2 mutants. Notably, UNC-40 and F-actin were no longer tightly localized to the invasive cell membrane of the AC in madd-2 mutants (Fig. 7, F and G). Furthermore, in contrast to the robust localization of F-actin along the invasive cell membrane in wild-type animals (n = 10/10 ACs; Fig. 8, A–C; and Video 5), time-lapse analyses revealed that madd-2 mutants often had transient, mislocalized patches of F-actin along the AC’s apical and lateral domains (n = 6/12 animals; Fig. 8, D–F; and Video 5). The specific function of MADD-2 in regulating UNC-6–independent UNC-40 clustering and the perturbation in UNC-40 polarization toward UNC-6 in madd-2 mutants offer compelling evidence that ligand-independent UNC-40 (DCC) clustering is required for robust polarization toward UNC-6 (netrin).